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ASD Patient-Derived Neural Stem Cells Exhibit Defective Proliferation in Comparison to Sibling Control
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by abnormalities in social interactions and stereotyped/restrictive behavior. The study of ASD etiology has been hindered by disease heterogeneity and difficulties in creating representative mouse models. To examine neurobiological deficits in idiopathic autism, we (NJ Autism Center of Excellence; PI Millonig) generated iPSC lines from 8 severely affected males with ASD and their unaffected brothers (Sib) and derived neural stem cells (NSCs) to study fundamental developmental processes.
Objectives: Our goal is to uncover potential differences in proliferation in idiopathic autism patient-derived NSCs by using developmentally relevant exogenous factors (EFs). We have examined an array of EFs including FGF, pituitary adenylate cyclase-activating peptide, BDNF, NT3, 5-HT, H2O2 and MeHg.
Methods:
To define effects, cells were grown at high density (50K cells/cm2) without and with EFs. At 48h cells were labeled with tritiated thymidine to assess DNA synthesis and EdU for S-phase entry. In parallel, single cell analyses were conducted by acutely dissociating high-density cultures, plating at low density (10K/cm2) and fixing at 2h for immunostaining. Further, sister cultures were dissociated every 48h for 6 days to quantify live cell numbers via hemocytometer. To address possible variability, studies were performed blind, and all results have been replicated in multiple NSCs obtained from 2-3 independent iPSC lines for each subject.
Results:
As might be expected, FGF induced increases in DNA synthesis and S-phase entry by ~50% at 48h. These early increases predict 50% – 100% increases in cell numbers by 4 and 6 days. Though both ASD and sibling NSCs show a response to FGF stimulation, ASD NSCs from a single-family comparison exhibit a remarkable proliferation defect under baseline conditions. At 48 hours ASD NSCS display a 50% reduction in DNA synthesis. Additionally, ASD NSCs exhibit a 33% reduction in the proportion of cells entering S-Phase as well as a 75% reduction in cell numbers after 6 days in culture. Furthermore, preliminary results suggest ASD NSCs have differential proliferative responses to growth regulator, 5-HT, and greater sensitivity to environmental toxicant, MeHg, in comparison with sibling control.
Conclusions:
In aggregate, our results indicate that we are able to employ EFs to discover differences in ASD-implicated biological processes, namely proliferation. Ultimately, this methodology may lead to the development of personalized pharmacological treatment for ASD individuals. Future studies will examine underlying mechanisms by assessing NSC cell death, cell cycle machinery, mRNA transcriptome, and metabolomic profiles.